1. Field of the Invention
The present invention relates generally to the field of electrogenerated chemiluminescence (ECL). More particularly, it concerns a multimetallic assembly with ligands that bridge independent chromophores for use in ECL devices and ECL methods of improved sensitivity.
2. Description of Related Art
Electrogenerated chemiluminescence, alternatively referred to as electrochemiluminescence, involves the formation of excited state species as a result of highly energetic electron-transfer reactions of reactants formed electrochemically. ECL systems and devices that make use of molecules that luminesce upon electrical excitation have been widely studied and are used for such purposes as display devices and instruments for chemical analysis. Several reviews have appeared on both the theory and application of ECL (Faulkner and Bard, 1977; Faulkner and Glass, 1982; Yang et al., 1994; Knight and Greenway, 1994).
The first report of ECL in a metal chelate appeared in 1972, in which the excited state of Ru(bpy)32+ was generated in nonaqueous media by electrochemical formation and subsequent annihilation of the reduced Ru(bpy)3+ and oxidized Ru(bpy)33+ species (Tokel and Bard, 1972).
Ru(bpy)32++exe2x88x92xe2x86x92Ru(bpy)3+xe2x80x83xe2x80x83(1)
Ru(bpy)32+xe2x88x92exe2x88x92xe2x86x92Ru(bpy)33+xe2x80x83xe2x80x83(2)
Ru(bpy)33++Ru(bpy)3+xe2x86x92Ru(bpy)32++Ru(bpy)32+*xe2x80x83xe2x80x83(3)
Ru(bpy)32+*xe2x86x92Ru(bpy)32++hvxe2x80x83xe2x80x83(4)
The potential range (window of stability) in nonaqueous solvents (e.g., +2.5 to xe2x88x922.5 V vs. NHE in MeCN) allows formation of the energetic precursors necessary in the annihilation sequence. However, given the limited potential window of water, alternative means must be used to produce the excited state (e.g., Ru(bpy)32+*) for aqueous ECL. For example, in the presence of a luminophore such as Ru(bpy)32+, oxidation of species like oxalate or tripropylamine (TPrA) or reduction of a species like peroxydisulfate (S2O82xe2x88x92) have been shown to generate the necessary energetic precursors for excited state formation (Yang et al., 1994; Knight and Greenway, 1994; Rubinstein and Bard, 1981; Rubinstein et al., 1983; Ege et al., 1984; White and Bard, 1982; McCord and Bard, 1991; Leland and Powell, 1990). The presumed mechanism involves formation of strong reductants (CO2xe2x88x92.or TPrA.) or strong oxidants (SO4xe2x88x92.) that can interact with Ru(bpy)33+ or Ru(bpy)3+ respectively, to produce the excited state:
Ru(bpy)33++TPrA.xe2x86x92Ru(bpy)32+*+productsxe2x80x83xe2x80x83(5)
or
Ru(bpy)3++SO4xe2x88x92.xe2x86x92Ru(bpy)32+*+SO42xe2x88x92xe2x80x83xe2x80x83(6)
Ru(bpy)32+ is used as an ECL-active label in DNA and immunoassay probes and for clinical analyses (U.S. Pat. Nos. 5,221,605; 5,238,808; 5,310,687; 5,453,356; 5,714,089; 5,731,147; Ege et al., 1984; Blackburn et al., 1991). ECL has several advantages over other detection techniques like fluorescence because no excitation source is required, and thus, ECL is immune to interference from luminescent impurities and scattered light. However, as with fluorescence labeling techniques, the sensitivity of the analysis depends on the ECL efficiency of the label.
With the goal of increasing the magnitude of ECL emission, this earlier work was extended to the use of multimetallic systems. Several reports on ECL with multimetallic systems have appeared, including Mo and W clusters (Mussel and Nocera, 1990; Ouyang et al., 1986) and a bimetallic Pt complex, Pt2(xcexcxe2x88x92P2O5H2)44xe2x88x92 (Vogler and Kunkeley, 1984; Kim et al., 1985). However, the ECL efficiency (taken as the number of photons emitted per redox event) in these systems was much weaker than Ru(bpy)32+ under the same experimental conditions. Moreover, these earlier studies precluded the use of water because of the insolubility and instability of these complexes in an aqueous environment (Mussel and Nocera, 1990: Ouyang et al., 1986; Vogler and Kunkeley, 1984; Kim et al., 1985).
There have been no reports of ECL in multimetallic ruthenium systems. Often, coordination of a second metal center through a bridging-ligand (BL) framework (e.g., L2M(BL)ML2) leads to decreased photoluminescence quantum efficiencies and excited-state lifetimes. For example, Ru(bpy)32+ has an excited-state lifetime of emission (xcfx84em) of about 600 ns (Bock et al., 1974; Bock et al., 1979; Navon and Sutin, 1974; Sutin and Creutz, 1978; Meyer, 1978; Hage et al., 1990; Barigelletti et al., 1991; and references therein; Demas and Crosby, 1971) and an emission quantum efficiency (xcfx86em) in MeCN of 0.086 (Kawanishi et al., 1984). Replacement of one bipyridine with a ligand capable of bridging two independent metal centers such as 2,3-bis(2xe2x80x2-pyrifyl)pyrazine (dpp) results in a decrease of xcfx86em to 0.064 for Ru(bpy)2(dpp)2+ and xcfx84emxcx9c200 ns. [In Brauenstein et al., 1984, Brauenstein reported the relative quantum efficiencies of Ru(bpy)2(dpp)2+ and [(bpy)2Ru]2(dpp)4+ compared to Os(bpy)32+ (0.0348xc2x10020) (Demas and Crosby, 1971). The values shown are scaled to Ru(bpy)32+ (xcfx84em=0.086) (Kawanishi et al., 1984) to make comparisons more valid.] Addition of a second Ru(bpy)22+ moiety to form [(bpy)2Ru]2(dpp)4+ gives xcfx86em=0.0007 and xcfx84em less than 50 ns (Brauenstein et al., 1984). This appears to be the general behavior. Other studies on Ru(II) diimine systems have shown that the monometallic parent complex might be luminescent in fluid solution at room temperature, but the bimetallic system is usually not (Dose and Wilson, 1978; Hunziker and Ludi, 1977; Goldsby and Meyer, 1984; Richardson et al., 1982; Richter and Brewer, 1993). A number of these systems were prepared in mixed oxidation states (i.e., L2MIII(BL)MIIL2) with the goal of defining the intervalence charge-transfer transition that is often present in the mixed-valence state (Creutz and Taube, 1969; Creuiz and Taube, 1972; Elias and Drago, 1972; Callahan et al., 1974; Callahan et al., 1975; Tom and Taube, 1975; Krentzien and Taube, 1976; Powers et al., 1976). In such studies, luminescence is not necessary to probe the photophysical and charge-transfer behavior. However, luminescence is a necessary prerequisite for efficient ECL.
The emission displayed by [(bpy)2Ru]2(dpp)4+ and its monometallic analogue in fluid solution at room temperature has been traced to the weak metal-metal interaction present in the bimetallic system and the bipyridine-like environment conferred by the bridging dpp ligand (Brauenstein et al., 1984). However, even in this case, luminescence in the bimetallic system is much weaker than that observed in the parent compound. Many photophysical studies on ruthenium and osmium multimetallic complexes have centered on systems where the degree of electronic coupling between metal centers, as mediated by the BL-based orbitals, varies over orders of magnitude (i.e., Robin and Day Class II and III systems) (Dose and Wilson, 1978; Hunziker and Ludi, 1977; Goldsby and Meyer, 1984; Richardson et al., 1982; Richter and Brewer, 1993; Creutz and Taube, 1969; Creutz and Taube, 1972; Elias and Drago, 1972; Callahan et al., 1974; Callahan et al., 1975; Tom and Taube, 1975; Krentzien and Taube, 1976; Powers et al., 1976; Robin and Day, 1967; Creutz, 1983). In such systems, increased electronic coupling between metal centers is directly influenced by the energy and density of states of the BL. Increasing electronic density on the lowest-unoccupied xcfx80* molecular orbitals and the acceptor orbitals active in the metal-to-ligand charge transfer (MLCT) transitions that produce the excited state leads to enhanced communication. However, such systems rarely display high photoluminescence efficiencies. In fact, these systems rarely display any photoluminescence in fluid solution (Dose and Wilson, 1978; Hunziker and Ludi, 1977; Goldsby and Meyer, 1984; Richardson et al., 1982; Richter and Brewer, 1993). Despite the wealth of data on systems with significant interaction between metal centers, much less has been done on those where there is very weak coupling so that the metal centers are isolated or valence trapped (Robin and Day Class I systems) (Robin and Day, 1967; Creutz, 1983).
In recent reports on bimetallic ruthenium systems with small electronic coupling between metal centers (Baba et al., 1995; Boyde et al., 1990; Liang et al., 1996), excited-state lifetimes that were greater than those for monometallic derivatives were reported. For example, [(dmb)2Ru]2(bbpe)4+ (Boyde et al., 1990) and [(dmb2Ru]2(bphb)4+ (Baba et al., 1995) [dmb =4,4xe2x80x2-dimethyl-2,2xe2x80x2-bipyridine, bbpe=trans-1,2-bis(4xe2x80x2-methyl-2,2xe2x80x2-bipyridyl-4-yl)ethene, and bphb=1,4-bis(pxe2x80x2-methyl-2,2xe2x80x2-bipyridin-4-yl)benzene] have xcfx84em=1.31 and 1.57 xcexcs, respectively, compared to 0.95 xcexcs for the tris-substituted Ru(dmb)32+ system. The monometallic species (dmb)2Ru(bphb)2+ yielded xcfx84em=1.34 xcexcs and xcfx86em=0.109; while the xcfx86em for [(dmb)2Ru]2(bphb)4+ was 0.125. Thus, in contrast to previously studied systems, these bimetallic complexes clearly show increased efficiencies and lifetimes over the monometallic ones. This has been attributed to a larger Ru(dxcfx80)xe2x86x92bphb(xcfx80*) transition dipole and a smaller electron-vibrational coupling constant, resulting in a smaller degree of excited state distortion (Baba et al., 1995; Boyde et al., 1990).
Provided herein is an efficient multimetallic ECL compound. The present invention is based upon a bimetallic ruthenium species wherein the ruthenium chromophores are electronically and spatially isolated via a bridging, chelating ligand. The compound is soluble in aqueous media and emits under similar conditions as Ru(bpy)32+ but with 2-3 times the magnitude of emission. The availability of labels with much higher luminescence sensitivity, as described herein, provides a marked improvement over currently available ECL labels by extending the useful range of ECL in analytical applications, particularly in DNA probe technology, where detection of biomolecules without resorting to amplification (e.g., PCR(trademark) amplification) is highly advantageous.
As used herein, the multimetallic compounds for use in ECL systems include at least two metal ions, preferably ruthenium or osmium, with ruthenium being the most preferred. The invention may also include chemical moieties having more than two metal centers. Each metal ion is surrounded with ligands such that the total number of bonds between the ligands and the metal ion equals the coordination number of the metal ion. The ligands may all be the same or each metal may be bonded to a number of different ligands. At least one of the ligands is a bridging ligand, having bonds to at least two of the metal ions.
Bridging ligands may include, but are not limited to, dpp, bbpe, and bphb, with bphb being the most preferred bridging ligand. Non-bridging ligands may include both monodentate and polydentate ligands. The ligands may be substituted with groups, including carboxylate esters, that may be used to conjugate the ECL compound to other molecules, such as antibodies, cells, polypeptides, nucleic acids, polysaccharides, steroids, alkaloids, non-biological polymers and the like for use in chemical and biochemical analysis. Ligands may also be substituted with hydrophilic or hydrophobic groups to modulate their solubility properties. The most preferred non-bridging ligands of the invention are substituted and unsubstituted 2,2xe2x80x2-bipyridine (bpy) groups. The most preferred compound of the invention is [(bpy)2Ru]2(bphb)4+.
In preferred embodiments of this invention, multimetallic ECL compounds are used in ECL cells and display devices. Because of their high ECL efficiencies, multimetallic systems of this type are particularly useful in the design of new labels for bioanalytical applications. Thus, in other preferred embodiments of the invention, the multimetallic ECL compounds are used as labels in systems for immunochemical analysis, DNA probes and in the detection of other biochemical and chemical compounds. The compounds may also be used in systems involving magnetic bead technology.
In one embodiment, the present invention is a chemical moiety, and a method of determining the presence of a chemical moiety, the method including (a) forming a reagent mixture containing the chemical moiety, or the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, the chemical moiety having the formula 
wherein Mxe2x80x2 and Mxe2x80x3 are independently selected from the group consisting of ruthenium and osmium; L1, L2, L3, and L4 each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of the substituted ligands is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, arnidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of the ligands being the same or not the same as each other ligand; P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, xcex1,xcfx89-(2,2xe2x80x2-bipyrid-4-yl)-alkanes), 4,4xe2x80x2-bipyridines, and 1,2-bis(diphenylphosphine)ethane; B is a biological substance, a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with a complementary material, or a non-biological polymer; t is an integer equal to or greater than 1; u is an integer equal to or greater than 1; the link being covalent bonding linking B with at least one of L1, L2, L3, and L4; L1, L2, L3, L4, P and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce; (b) exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that the chemical moiety is oxidized and the agent forms a reductant, or such that the chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and (c) detecting emitted luminescence thereby to determine the presence of the chemical moiety.
In certain aspects, the link is one or more amide linkages, ester or thioester linkages, or ether or thioether linkages, each of which linkages covalently bonds B with one of L1, L2, L3, and L4. In other aspects, B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer. In certain specific aspects, B is a serum-derived antibody or a monoclonal antibody, a nucleotide or polynucleotide, or a T4 thyroid hormone.
Other embodiments of the present invention include a method of determining the presence of an analyte of interest which binds to a chemical moiety, the moiety having a having a formula as described hereinabove, and the method including the steps of (a) forming a reagent mixture including the chemical moiety, or including the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, and the analyte of interest; such that the chemical moiety and the analyte specifically bind to one another; (b)exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that the chemical moiety is oxidized and the agent forms a reductant, or such that the chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and (c) detecting emitting luminescence thereby to determine the presence of the analyte of interest.
In certain aspects of the invention, the analyte is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer. In certain specific aspects, the analyte is insulin, digoxin, digitoxin, T4 thyroid hormone, a fungus, an antibody, a pharmacological agent, or sugar.
In yet other embodiments, the invention includes a competitive binding method of determining the presence of an analyte of interest wherein the analyte and a chemical moiety bind competitively to a complementary material, the method including (a) forming a reagent mixture including the analyte of interest, the complementary material and the chemical moiety, or the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, such that the chemical moiety and the analyte of interest bind competitively to the complementary material; (b) exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that the chemical moiety is oxidized and the agent forms a reductant, or such that the chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and (c) detecting emitted luminescence thereby to determine the presence of the analyte of interest.
In additional embodiments, the present invention includes a system for determining the presence of a chemical moiety, the system including (a) a reagent mixture including the chemical moiety, or the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant; (b) means for exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that the chemical moiety is oxidized and the agent forms a reductant, or such that the chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and (c) means for detecting emitted luminescence thereby to determine the presence of the chemical moiety.
In another embodiment, the invention includes a system for determining the presence of an analyte of interest which binds to a chemical moiety, the system including (a) a reagent mixture including the chemical moiety, or including the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, and the analyte of interest; (b)means for contacting the chemical moiety with the analyte of interest to form a reagent mixture; (c) means for exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that the chemical moiety is oxidized and the agent forms a reductant, or such that the chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and (d) means for detecting emitted luminescence thereby to determine the presence of the chemical moiety.
In one embodiment, the present invention includes a chemical moiety and a method of determining the presence of the chemical moiety having the formula:
[(L1)n(L2)o(L3)p(L4)q(L5)r(L6)sMxe2x80x2(P)Mxe2x80x3(L7)v(L8)w(L9)x(L10)y(L11)z(L12)k]t(B)u
wherein Mxe2x80x2 and Mxe2x80x3 are independently selected from ruthenium and osmium; L1, L2, L3, L4, L5, and L6 are ligands of Mxe2x80x2, each of which may be the same as or not the same as each other ligand; L7, L8, L9, L10, L11, and L12 are ligands of Mxe2x80x3, each of which may be the same as or not the same as each other ligand; P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, xcex1,xcfx89-(2,2xe2x80x2-bipyrid-4-yl)-alkanes), 4,4xe2x80x2-bipyridines, and 1,2-bis(diphenylphosphine)ethane; B is a substance which is attached to one or more of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, and L12; each of n, o, p, q, r, s, v, w, x, y, z, and k is zero or an integer; t is an integer equal to or greater than 1; u is an integer equal to or greater than 1; L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, P and B being of such composition and number that the chemical moiety can be induced to electrochemiluminesce and the total number of bonds to Mxe2x80x2 provided by the ligands of Mxe2x80x2 equals the coordination number of Mxe2x80x2, and ; and the total number of bonds to Mxe2x80x3 provided by the ligands of Mxe2x80x3 equals the coordination number of Mxe2x80x2; the method including (a) forming a reagent mixture under suitable conditions containing the chemical moiety; (b) inducing the chemical moiety to electrochemiluminesce by exposing the reagent mixture to electrochemical energy; and (c) detecting emitted luminescence and thereby determining the presence of the chemical moiety. In certain aspects, the invention B may be a substance covalently bound to one or more of L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, or L12 through one or more amide linkages.
In other embodiments, the invention includes a method of determining the presence of an analyte of interest which binds to a chemical moiety, the chemical moiety having the formula:
[(L1)n(L2)o(L3)p(L4)q(L5)r(L6)sMxe2x80x2(P)Mxe2x80x3(L7)v(L8)w(L9)x(L10)y(L11)z(L12)k]t(B)u
the method including (a) contacting the analyte with the chemical moiety under suitable conditions so as to form a reagent mixture such that the B substance of the chemical moiety and the analyte are capable of binding with one another; (b) inducing the chemical moiety to electrochemiluminesce by exposing the reagent mixture to electrochemical energy; and (c) detecting emitted luminescence and thereby determining the presence of the analyte of interest.
In yet other embodiments, the invention includes a competitive binding method of determining the presence of an analyte of interest wherein the analyte and a chemical moiety bind competitively to a chemical material, the chemical moiety having the formula:
[(L1)n(L2)o(L3)p(L4)q(L5)r(L6)sMxe2x80x2(P)Mxe2x80x3(L7)v(L8)w(L9)x(L10)y(L11)z(L12)k]t(B)u
the method including: (a) binding the material, the B substance of the chemical moiety and the analyte under suitable conditions such that the chemical moiety and the analyte are capable of competitively binding with the material so as to form a reagent mixture containing the chemical moiety; (b) inducing the chemical moiety to electrochemiluminesce by exposing the reagent mixture to electrochemical energy; and (c) detecting emitted luminescence and thereby determining the analyte of interest.
In certain aspects, B is the same substance as the analyte. In certain other aspects, the material is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or amino acid. In specific aspects, the chemical material is a serum-derived antibody or a monoclonal antibody, a DNA or RNA fragment, a pharmacological agent or sugar. The method may be a competitive binding method wherein the material is fixed to an insoluble matrix. In one aspect, the invention includes a method further defined as a heterogeneous method wherein the material is a monoclonal antibody and the insoluble matrix is the surface of an assay vessel. In another aspect, the invention includes a homogeneous method wherein the material is a monoclonal antibody and the insoluble matrix is the surface of an assay vessel.
In another embodiment, the invention includes a system for determining the presence of a chemical moiety having the formula:
[(L1)n(L2)o(L3)p(L4)q(L5)r(L6)sMxe2x80x2(P)Mxe2x80x3(L7)v(L8)w(L9)x(L10)y(L11)z(L12)k]t(B)u
the system including (a)a reagent mixture including the chemical moiety; (b) means for inducing the chemical moiety to electrochemiluminesce; and (c) means for detecting emitted luminescence. In certain aspects of the system, the reagent mixture also includes one or more different chemical moieties each of which can be induced to luminesce at a different wavelength. In certain other aspects of the system, the reagent mixture also includes one or more different chemical moieties each of which can be induced to luminesce by exposure to energy of a different value or from a different source.
In yet another embodiment, the invention includes a system for determining the presence of an analyte of interest which binds to a chemical moiety, the moiety having the structural formula:
[(L1)n(L2)o(L3)p(L4)q(L5)r(L6)sMxe2x80x2(P)Mxe2x80x3(L7)v(L8)w(L9)x(L10)y(L11)z(L12)k]t(B)u
the system including (a) the chemical moiety; (b) a means for contacting the chemical moiety with the analyte of interest to form a reagent mixture such that the B substance of the chemical moiety and the analyte are capable of binding with one another; c) a means for inducing the chemical moiety to electrochemiluminesce; and d) a means for detecting emitted luminescence. In certain aspects of the system, the reagent mixture includes one or more different chemical moieties each of which can be induced to luminesce at a different wavelength, each moiety being attached to a different analyte of interest. In certain other aspects of the system, the reagent mixture includes one or more different chemical moieties each of which can be induced to luminesce by exposure to energy of a different value or from a different source, each moiety being bound to a different analyte of interest.